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Published ahead of print on November 11, 2005, doi:10.1165/rcmb.2005-0191OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 348-354, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0191OC

T Cell Chemotaxis and Chemokine Release after Staphylococcus aureus Interaction with Polarized Airway Epithelium

Sandie Escotte, Denise Al Alam, Richard Le Naour, Edith Puchelle, Moncef Guenounou and Sophie C. Gangloff

Laboratoire d'Immuno-Pharmacologie Cellulaire et Moléculaire, EA3796, Université de Reims Champagne Ardennes, IFR53; and INSERM UMRS 514, IFR 53, Reims, France

Correspondence and requests for reprints should be addressed to Prof. S. C. Gangloff, Laboratoire d'Immuno-pharmacologie cellulaire et moléculaire, EA3796-IFR53, 1 Avenue du Maréchal Juin, 51100 Reims, France. E-mail: sophie.gangloff{at}univ-reims.fr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In response to bacterial infection, airway epithelium releases inflammatory mediators including cytokines and chemokines that lead to immune cell efflux and could stimulate the adaptive T cell immune response. The aim of our study was to analyze, in a double chamber culture, the chemokine changes in response to Staphylococcus aureus and their consequences for T cells. Our data show that S. aureus stimulates basolateral and apical release of IL-8 and eotaxin by airway epithelial cells. We also observed increased chemokine receptor expression on CD8+ and CD4+ T cells and enhanced chemotaxis of CD4+ T cells toward apical supernatant. Our data strongly suggest that S. aureus interaction with airway epithelium contributes to specific migration of T cells to inflamed sites.

Key Words: airway epithelial cells • chemokines • chemotaxis • Staphylococcus aureus • T lymphocytes


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Airway epithelium, which is exposed to many inhaled particles, is a mechanical barrier that separates the submucosal environment from the lumen milieu. Airway epithelial cells are actively involved in the innate and acquired immune response and in the pathogenesis of airway inflammation. In response to bacterial infection, airway epithelium releases a broad spectrum of factors, including antibacterial factors, cytokines, and chemokines, that modulate the inflammatory response (1). Chemokines and their receptors are important factors underlying the mechanisms regulating the tissue-specific immune cell recruitment. Several chemokines are produced either constitutively or in the setting of airway inflammation. In humans, several airway infection studies have reported increased expression of IL-8 (2, 3), eotaxin (4, 5), regulated upon activation, normal T cell expressed and secreted (RANTES) (6) and IFN-{gamma}–inducible protein-10 (IP-10) (7). The CXC chemokines IL-8 and IP-10 are chemotactic for neutrophils, monocytes, and T lymphocytes. Eotaxin and RANTES, CC chemokines, are also chemotactic for inflammatory cells like eosinophils, basophils, mast cells, monocytes, and lymphocytes (8). Furthermore, selective homing and/or preferential accumulation of these effector cells are also regulated by the expression of a specific chemokine receptor pattern on their surface (9–11).

T cells are critical adaptative mediators in inflammatory diseases, endowed with the capacity to initiate, amplify, and terminate antigen-specific immune responses. They are a highly heterogeneous population that shows complex patterns of trafficking (e.g., presence of specific CD4+ or CD8+ T cell subsets in the infectious environment) (12, 13). Type 1 and type 2 T cells can be functionally identified among the CD4+ and CD8+ T cells on the basis of their cytokine pattern and their chemokine receptor pattern (14). A number of pulmonary diseases are characterized by preferential accumulation of type 1 or type 2 T cells, which specifically modulate defined aspects of pathogenesis in affected tissues (15, 16). For example, T cells infiltrating the airway of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis were shown to have a type 1 profile and to express high levels of CXCR3, the IP-10 receptor (17). T cells isolated from asthmatic human bronchoalveolar lavage fluid were found to have a type 2 profile and to express preferentially CCR5 and CXCR3, the receptors for RANTES and IP-10, respectively (18). The selective recruitment of type 1 or type 2 T cells into the lung is therefore a critical event for the development of the pathogenesis of airway inflammatory diseases.

Staphylococcus aureus, classically considered as an extracellular pyogenic pathogen that can persist in epithelia, is one of the first pathogens to colonize the respiratory tract. S. aureus infections are common in patients with compromised airway defense as in cystic fibrosis (CF) or nosocomial pulmonary infection. Upon interaction with the epithelium, the bacterium is known to induce antimicrobial activity (19), cell apoptosis (20), production of proinflammatory mediators like IL-8 and IL-6 (21), and to stimulate T cell activation via superantigen (22). Also, the host response to S. aureus is dominated by polymorphonuclear leukocytes (PMN). However, little is known about the consequences of the early airway epithelium–S. aureus interaction for T cell mobilization and polarization.

In the present study, we used a polarized human MM-39 tracheal cell model retaining serous secretory functions to study the effects of live S. aureus on respiratory epithelium and the consequences for circulating T cells. Our data show that S. aureus stimulates significantly apical and/or basolateral release of IL-8, eotaxin, and RANTES by airway epithelial cells. Moreover, we demonstrate that S. aureus interaction with airway epithelial cells also induces a specific chemokine receptor pattern on the T cell subpopulation and an enhanced chemotaxis of CD4+ T cells toward apical airway epithelial cell supernatant. CD4+ T cells exhibited increased expression of the IL-8 and IP-10 receptors, while CD8+ T cells showed increased expression of the IL-8 and eotaxin/RANTES receptors. Taken together, our data suggest that after 3 h interaction with airway epithelium, live S. aureus induces significant changes in both apical and basolateral cell secretions, leading to specific T cell accumulation at the inflamed site.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Bacterial Strain and Growth Conditions
S. aureus strain 8325–4, a wild-type laboratory strain (NCTC 8325 cured of prophages), was a generous gift from T. J. Foster (Department of Microbiology, Trinity College, Dublin, Ireland). Bacteria were stored at –20°C in trypticase soy broth (TSB-bioMérieux S.A., Marcy l'Etoile, France) containing 20% (vol/vol) glycerol. Before each assay, bacteria were cultured in trypticase soy broth (TSB) at 37°C with shaking up to stationary growth phase. An aliquot of the culture was then inoculated into fresh TSB at a starting OD{lambda}=600nm = 0.1 and cultured at 37°C up to an OD{lambda}=600nm = 0.6. In these conditions, bacteria used for the interaction with epithelial cells were in their exponential growth phase.

Airway Epithelial Cell Culture and Interaction with S. aureus
A transformed human tracheal gland cell line MM-39, developed by M. Merten (EMI 0014 Inserm, Vandoeuvre-les-Nancy, France), was cultured as previously described (23). Briefly, cells were grown at 37°C under 5% CO2 atmosphere on type I collagen–coated flasks in Dulbecco's modified Eagle's medium (DMEM) F-12 mixture (Invitrogen, Cergy-Pontoise, France) supplemented with 1% Ultroser G serum substitute (Biosepra, Villeneuve La Garenne, France), glucose (10 g/liter), sodium pyruvate (0.33 g/liter), penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin B (2 µg/ml). Polarized monolayers of MM-39 were prepared by seeding 5 x 105 cells/cm2 on a Transwell (3 µm, 1.1-cm2, polyester; Becton Dickinson Labware, Franklin Lakes, NJ) (Figure 1A). The cells were maintained at 37°C under 5% CO2 atmosphere, in a liquid:liquid system with 500 µl supplemented DMEM F-12 in the apical compartment and 700 µl supplemented DMEM F-12 in the basolateral compartment. After 8–10 d of culture, airway epithelial cells were confluent (Figure 1B), and the formation of tight junctions was functionally assessed by measurements of the electrical resistance across monolayers. The uninfected monolayer steady-state resistance of 300 {Omega}/cm2 was reached in 8–9 d. Airway epithelial cells showed a glandular serous type with a polarized secretory leukoprotease inhibitor (SLPI) secretion (a serous cell–specific secretory marker) (Figure 1C). Twelve hours before the interaction with bacteria, the cell culture media were replaced with fresh DMEM F-12 medium, and the media were changed once again just before the experiment. For each experiment, cultures were apically infected with live S. aureus, at a ratio of 30 bacteria for 1 epithelial cell (3.3 x 107 cfu/ml), for 3 h. Epithelial integrity was confirmed by the measurement of the transepithelial resistance, which remained constant through the 3 h of interaction. At the end of the interaction, the apical (500 µl) and the basolateral (700 µl) supernatants were collected. To obtain cell-free supernatants and to normalize the volume of the apical versus basolateral supernatant, 200 µl DMEM F-12 were added to the apical supernatant; the supernatants were then centrifuged (10,000 rpm, 10 min, 4°C) and passed through a 0.45-µm membrane filter (Millipore, Molsheim, France).


Figure 1
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  		Figure 1. Airway epithelial cell double chamber culture model. (A) Airway epithelial cells were grown on the upper side of porous polycarbonate filters coated with collagen I. (B) Confluent airway epithelial cells (MM-39) (transversal section and phase-contrast microscopy) after 10 d of culture. (C) Apical and basolateral secretion of SLPI, a serous glandular cell-specific marker. Bar, 10 µm. Original magnification: x400, ***P < 0.001 compared with basolateral supernatants.

 
Isolation and Culture of Human Peripheral Blood Lymphocytes
Enriched human peripheral blood lymphocyte preparations from healthy consenting donors, after protocol approval by the Ethics Committee of the CHU de Reims, were obtained by counter-courant elutriation (Department of Haematology, CHU de Reims). Lymphocytes were prepared by standard density gradient centrifugation (Lymphoprep, Abcys, Paris, France). T cell subsets were purified by immunomagnetic negative selection using the pan T cell biotin-antibody cocktail and the human anti-CD4 and anti-CD8 mAb (microbeads MACS; Miltenyi, Paris, France). Purity of CD3+ lymphocyte, CD4+ T lymphocyte, and CD8+ T lymphocyte preparations was in the range of 81.1–99.5%, as assessed by flow cytometry with human CD19, CD3, CD8, and CD4 monoclonal antibody (mAb) (BD Biosciences, San Diego, CA). Lymphocytes were cultured in RPMI 1640 (Invitrogen) containing L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% filtered heat-inactivated FCS (Invitrogen) and resuspended at a concentration of 1 x 106 cells/ml.

Chemotaxis Assay
Experiments were performed on the 1.1-cm2, 3-µm pore size polyester filters (Becton Dickinson Labware). Purified T lymphocytes (6.104 cells) were resuspended in DMEM F-12 and loaded in the upper chamber. Different airway epithelial cell supernatants (AECS) were loaded in the lower chamber. After 24 h incubation at 37°C under 5% CO2, T cells present in the lower chamber were counted. To compare the results from different experiments, a chemotaxis index (CI = number of T cells migrating toward the epithelium supernatant/number of T cells migrating toward control media) was calculated. The number of cells represents the mean of five high-power fields for each condition. The results are reported as the mean CI ± SEM of at least three independent experiments.

For the neutralizing IL-8 chemotaxis assay, the epithelial cell supernatants were preincubated with anti–IL-8 antibodies (R&D Systems, Lille, France). Enough antibody was used to inhibit responses to 10 times more human recombinant IL-8 than the quantity of IL-8 found in the different epithelial supernatants (Table 1).


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  		TABLE 1. LYMPHOCYTE CHEMOTAXIS INDEX IN RESPONSE TO IL-8

 
Chemotaxis experiments on solidified 2% agarose (Sigma Chemical Corp., St. Louis, MO) 35-mm Petri dishes (5 ml/well) were also performed. Briefly, three equidistant 3-mm holes were punched in the agarose. Subsequently, the lymphocytes (1.106 cells) were loaded in the center hole and the cell culture supernatants or control media were added to each side hole. Petri dishes were incubated at 37°C in a humid chamber for 24 h, then fixed with a paraformaldehyde solution (4% in PBS), rinsed, and air dried. The distances were measured between the center of the hole and the edge of the rocket formed by the lymphocytes migrating toward cell supernatant (distance toward supernatant or DTS) or toward control media (distance toward medium or DTM) (Figure 2). The lymphocyte distance chemotaxis index (DCI = DTS/DTM) was then calculated. The results are reported as the mean DCI ± SEM of at least six independent experiments.


Figure 2
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  		Figure 2. Qualitative underagarose chemotaxis assay. Lymphocytes were loaded in the center hole (black circle). After 24 h, migration occurred toward apical supernatants of S. aureus–stimulated airway epithelial cells. The distances were measured between the center of the hole and the edge of the rocket formed by the lymphocytes migrating toward cell supernatants (DTS) or toward culture medium (DTM). Lymphocyte distance chemotaxis index (DCI = DTS/DTM) was then calculated.

 
Flow Cytometric Analysis
Reagents were from Becton Dickinson (CD19-FITC, CD3-PE, CD4-FITC, CD8-FITC, CXCR1-biotin, Streptavidin-PerCp, CXCR3-PE, and CCR3-PE). Enriched peripheral blood lymphocytes were stained according to the manufacturer's specifications for analysis by flow cytometry. For each sample, 0.5–1.106 cells were stained; comparable numbers of cells were used for each independent test. All data were obtained on FACScalibur and analyzed with CellQuest software (Becton Dickinson, San Jose, CA). At least 10,000 gated events were evaluated for each condition. Appropriate mAb isotypic controls were included in each analysis, with acquisition and analysis gates set accordingly.

ELISA of SLPI, IL-8, IP-10, Eotaxin, and RANTES
SLPI, IL-8, IP-10, eotaxin, and RANTES in airway epithelial cell culture supernatants were quantified by ELISA (Duoset and Quantikine; R&D Systems) following the manufacturer's instructions. The detection limit of each ELISA was 25 pg/ml, 25 pg/ml, 5 pg/ml, 1 pg/ml, and 1 pg/ml for SLPI, IL-8, IP-10, eotaxin, and RANTES, respectively.

Statistical Analysis
All data are presented as means ± SEM of at least three independent experiments. Statistical significance was determined by ANOVA (StatView Software, Cary, NC).


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
S. aureus Induces Polarized Secretion of Chemokines by Airway Epithelial Cells
To determine whether apical S. aureus interaction with polarized airway MM-39 epithelial cells induces the secretion of chemokines, the levels of IL-8, RANTES, eotaxin, and IP-10 were measured, by ELISA, in apical and basolateral AECS. In the absence of S. aureus, significant differences were observed in the apical supernatant versus basolateral supernatant (317.2 ± 96.8 pg/ml and 197.8 ± 81.6 pg/ml for IL-8 [P < 0.05]; 10.5 ± 4.0 pg/ml and 4.1 ± 2.5 pg/ml for RANTES, respectively). IP-10 release was similar in apical and basolateral AECS (19.2 ± 7.7 pg/ml versus 11.3 ± 4.5 pg/ml, respectively). Under our experimental conditions, eotaxin release was not detected (Figure 3). Exposure of epithelial cells to S. aureus resulted in drastic changes. IL-8 release increased significantly in both apical and basolateral supernatants (5.2-fold, P < 0.01; 2.6-fold, P < 0.05, respectively) as compared with control AECS. A significant increase of eotaxin in both supernatants (P < 0.01) was observed. In contrast, no significant changes in IP-10 and RANTES were observed after addition of S. aureus (Figure 3).


Figure 3
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  		Figure 3. Chemokine secretion by airway epithelial cells after apical S. aureus stimulation. Airway epithelial cells grown in the double chamber model were in contact with culture medium (–) or exposed to S. aureus (+). Apical (filled bars) and basolateral (open bars) cell culture supernatants were harvested after a 3-h interaction. IL-8, RANTES, eotaxin, and IP-10 were measured by ELISA. Results are the mean ± SEM of at least three independent experiments. (*P < 0.05, **P < 0.01). ND: not detected, under the detection limit.

 
S. aureus Interaction with Polarized Airway Epithelial Cells Enhances T Lymphocyte Chemotaxis
To test whether supernatants from S. aureus–stimulated epithelial cells affect the chemotaxis of circulating T lymphocytes, quantitative and qualitative migratory assays were performed. As shown in Figure 4, T cell contact with apical AECS led to a higher lymphocyte migration than contact with basolateral AECS when epithelial cells were not stimulated (3.0-fold versus 1.7-fold, respectively, compared with passive migration, P < 0.05) or S. aureus–stimulated (5.2-fold versus 3.7-fold, respectively, compared with passive migration, P < 0.01). It is of note that S. aureus–stimulated AECS always induced greater lymphocyte chemotaxis than nonstimulated AECS (P < 0.01). These data were double confirmed with the underagarose chemotaxis assay (Table 2).


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  		TABLE 2. LYMPHOCYTE DISTANCE CHEMOTAXIS INDEX IN RESPONSE TO SUPERNATANTS OF S. aureus–STIMULATED AIRWAY CELLS

 

Figure 4
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  		Figure 4. Lymphocyte migration toward supernatants of airway epithelial cells. Lymphocytes were incubated for 24 h with apical (filled bars) or basolateral (open bars) airway epithelial cell supernatant conditioned by the absence (–) or presence (+) of S. aureus. All data were expressed as a chemotaxis index (CI = number of T cells migrating toward the epithelium supernatant/number of T cells migrating toward control media). Results are the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 compared with culture medium.

 
To determine which T lymphocyte subsets migrated in response to supernatants from S. aureus–stimulated epithelial cells, purified CD8+ and CD4+ T lymphocytes were tested for chemotaxis. As shown in Figure 5A, in nonstimulated conditions, CD8+ T cell chemotaxis toward apical and basolateral supernatant was similar. CD4+ T cells migrated, however, preferentially toward the apical supernatant (2.7-fold for apical versus 1.5-fold for basolateral as compared with passive migration). After contact with S. aureus–stimulated AECS, the increase in the CD8+ T cell chemotaxis index (2.5-fold as compared with passive migration) was only observed when the cells were in contact with the apical supernatant. The CD4+ T cell chemotaxis was altered more, with impressive chemotaxis when these cells were in contact with the apical S. aureus–stimulated AECS (9.2-fold for stimulated versus 2.7-fold for nonstimulated) and to a lower extent when they were in contact with the basolateral S. aureus–stimulated AECS (2-fold for stimulated versus 1.5-fold for nonstimulated). When the chemotaxis of the two populations was compared, CD4+ T cells always exhibited a higher chemotaxis index than did CD8+ T cells (P < 0.05).


Figure 5
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  		Figure 5. T lymphocyte subset migration toward airway epithelial cell supernatants (A) and IL-8 involvement in T cell chemotaxis (B). (A) Apical supernatants of S. aureus–stimulated airway epithelial cells enhance chemotaxis of lymphocyte subsets. CD8+ (white bars) and CD4+ (gray bars) T cells were incubated for 24 h with airway apical or basolateral epithelial cell supernatant conditioned by the absence (–) or presence (+) of S. aureus. Data were expressed as a chemotaxis index. This index corresponds to the number of cells migrating toward supernatant/number of cells migrating toward culture medium. (B) Neutralization of IL-8 inhibits the migration of CD8+ and CD4+ T cells toward apical or basolateral supernatants of S. aureus–stimulated airway epithelial cells. CD8+ (white bars) and CD4+ (gray bars) T cells were incubated for 24 h with apical or basolateral supernatants of S. aureus–stimulated airway epithelial cells in the absence (–) or presence (+) of 1 µg/ml of IL-8 neutralizing antibody. Data were expressed as the percentage of the chemotaxis index. Results are the mean ± SEM of four independent experiments (*P < 0.05).

 
The increased IL-8 production by epithelial cells and enhanced T cell migration after contact with S. aureus–stimulated AECS suggested that IL-8 is a major chemoattractant for T cells. Indeed, complete inhibition of IL-8 function by anti–IL-8 antibodies showed an important effect of IL-8 on chemotaxis of CD8+ and CD4+ T cells toward both apical and basolateral AECS (Figure 5B). For CD8+ T cells, chemotaxis inhibition represented 55% and 50% in the apical and basolateral supernatants, respectively. For CD4+ T cells, the inhibition represented 40% in the apical supernatant and up to 90% in the basolateral supernatant.

Regulation of CXCR1, CXCR3, and CCR3 on the CD8+ and CD4+ T Cell Subsets in Contact with Supernatants of S. aureus–Stimulated Airway Epithelial Cells
To examine further the effects of the changes in the S. aureus–conditioned AECS on T cells, expression of the chemokine receptors CXCR1 (IL-8 receptor), CCR3 (RANTES and eotaxin receptor), and CXCR3 (IP-10 receptor) on the CD8+ and CD4+ T lymphocytes was analyzed. As shown in Figure 6A, CD8+ and CD4+ T cells treated with apical nonstimulated AECS expressed CXCR1 as well as CXCR3 and CCR3 on their surfaces. These receptor expressions were enhanced when the T cells were in contact with apical S. aureus–stimulated AECS. The CXCR1 and CCR3 expressions were increased on CD8+ T cells, and the CXCR1 and CXCR3 expressions were increased on CD4+ T cells. In contact with basolateral nonstimulated and S. aureus–stimulated AECS, only CXCR1 was expressed on CD4+ and CD8+ T cells (Figure 6B).


Figure 6
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  		Figure 6. Chemokine receptor expression on T cell subsets. Apical (A) and basolateral (B) supernatants of S. aureus–stimulated airway epithelial cell upregulates the expression of CXCR1, CXCR3, and CCR3 on lymphocyte subsets. Flow cytometric analysis of lymphocyte subsets after contact with apical or basolateral supernatants of S. aureus–stimulated versus nonstimulated airway epithelial cell supernatants was done with control nonbinding anti-isotype antibody and human CXCR1, CXCR3, and CCR3 monoclonal antibody. Lymphocyte subsets were stimulated for 3 h at 37°C. Dot plots shown represent one of at least three independent experiments. For each profile, the filled histogram represents the control isotype, the thin line represents T cells in contact with unstimulated airway epithelial cell supernatants, and the bold line represents T cells in contact with supernatants of S. aureus–stimulated airway epithelial cells.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Bacterial infections and inflammatory diseases involving surfaces lined by polarized epithelia often lead to excessive host responses with the presence of inflammatory cell infiltrates (24–27). In airway diseases like CF, the presence of leukocytes, such as neutrophils and T cells, in the bronchoalveolar lavages suggests that they are involved in host defense mechanisms and/or pathogenesis. The infiltration of these inflammatory cells is regulated by a large cytokine and chemokine network, where airway epithelial cell secretions have an important role in the establishment of an early signaling system for the host immune response. Beside the fact that it is established that IL-8 is involved in PMN attraction (26, 28, 29), T cells are found in the lumen in most airway diseases where IL-8 or eotaxin levels are increased, suggesting that these chemokines could play a role in T cell accumulation. While T cell trafficking into airway tissue plays a critical role in coordinating the immune response to infectious pathogens, the signals responsible for their migration are still not completely determined. The data presented here show that S. aureus, a major human respiratory pathogen, rapidly induces an enhanced polarized IL-8 gradient by the airway epithelial cells. They also show, for the first time, that the airway epithelial cells are able to produce RANTES, IP-10, and eotaxin. More importantly, the airway cell secretion alterations after cell contact with S. aureus have an impact on T cell chemokine receptor pattern and T cell trafficking.

Three-hour contact of live S. aureus with the apical side of the epithelial cells modulates chemokine secretions. Among the chemokines, IL-8 release was the most altered, with a 5-fold increase in the apical S. aureus–stimulated AECS as compared with the nonstimulated AECS. This result is in agreement with the data reported by Ratner and coworkers (21) and by Moreilhon and colleagues (30). Moreover, our double-chamber model allowed us to observe an increased IL-8 secretion in the basolateral compartment; this increase was always lower than the increase of IL-8 secretion in the apical compartment, leading therefore to a marked basolateral to apical gradient of IL-8 when live S. aureus were in contact with the airway epithelial cells. We also showed for the first time that S. aureus interaction with the airway epithelial cells accentuated the secretion of RANTES, eotaxin, and to a lesser extent of IP-10. The quantities of eotaxin, RANTES, and IP-10 produced by the airway epithelial cells were low but still within a range that is compatible with the chemotactic effects seen with purified chemokines (31).

S. aureus is known to induce the chemotaxis of PMNs and macrophages into the airway lumen. To our knowledge, the effects of S. aureus–airway epithelial cell interaction on T cell behavior have never been reported. Our study demonstrates that human purified circulating T cells were attracted by both apical and basolateral S. aureus–stimulated AECS and that the CD4+ T cells were the most reactive. This was clearly observed in the presence of the apical S. aureus–stimulated AECS, as the CD4+ T cell chemotaxis index increased up to 3-fold as compared with the nonstimulated apical AECS. Furthermore, IL-8 neutralization in the airway cell supernatants shows a predominant participation of this chemokine in T cell chemotaxis toward basolateral AECS (from 50% to 90%). This suggests that the increase in basolateral IL-8 release, due to the apical interaction of S. aureus with the airway epithelium, might be the main factor that directs CD4+ and CD8+ T cell migration toward the subepithelial inflamed area. Interestingly, when T cells are in contact with the apical S. aureus–stimulated AECS, IL-8 involvement in the migration process is reduced to 40%, suggesting that other factors play a role in the accumulation of T cells in the lumen. The effect of S. aureus–stimulated AECS on T cell CXCR1 (IL-8 receptor), CXCR3 (IP-10 receptor), and CCR3 (eotaxin and RANTES receptor) expression patterns was analyzed simultaneously with T cell chemotaxis. After 3 h contact with the basolateral S. aureus–stimulated AECS, only CXCR1 was detected at both the CD8+ and CD4+ T cell surfaces. This is in accordance with the fact that no significant difference was noted between the chemotaxis indices of CD4+ and CD8+ T cells, and that IL-8 is the major chemoattractant in these supernatants. However, when the T cells were pretreated for 3 h with the basolateral conditioned supernatants, they exhibited accentuated chemotaxis, suggesting that some, if not all, the cells became more sensitive to the chemoattractant in the basolateral media (data not shown). After 3 h of contact with the apical supernatants, a rapid and similar upregulation of CXCR1 on both CD4+ and CD8+ subsets was observed. This overexpression of the IL-8 receptor on T cells and the increase in IL-8 secretion by the airway cells could explain the augmentation in the CD4+ and CD8+ T cell chemotaxis indices in the presence of apical supernatants. Nevertheless, only 40% of this chemotaxis involves IL-8. The increased release of RANTES, eotaxin and IP-10 in the apical supernatants of S. aureus–stimulated airway cells and the specific upregulation of CXCR3 and CCR3 on CD4+ and CD8+ T cells, respectively, could explain the progressively reduced IL-8 chemoattractant potency and the distinct migration capacity of CD4+ and CD8+ T cells toward the airway cell media. This suggests that during their contact with the supernatants of S. aureus–stimulated airway epithelial cells, CD4+ and CD8+ T cells modify their surface chemokine receptor pattern and their capacity to migrate toward the airway cell media.

Chemokine receptor expression and migration of T cells have been studied mostly in models of endothelial or intestinal epithelial interaction with gram-negative or gram-positive bacteria (32–36). Our study clearly suggests that during an early S. aureus infection, before any disruption of the airway epithelium, IL-8 released on both sides of the airway epithelium is actively involved in the chemotaxis of circulating CD4+ and CD8+ T cells. In support of this, supernatants of S. aureus–stimulated airway epithelial cells upregulate not only CXCR1 on all T cells, but also CXCR3 on CD4+ T cells and CCR3 on CD8+ T cells, allowing cell specific migration toward the infectious site. Finally, our results clearly demonstrate that S. aureus infection of the airway epithelium is a key factor in recruitment of not only PMN but also lymphocytes in airways.


   Acknowledgments
 
The authors thank E. Caliot and Dr. E. Pringault (Laboratoire des interactions lympho-épithéliales, Institut Pasteur, Paris) for helpful suggestions on the double chamber model, Dr. T. J. Foster (Department of Microbiology, Trinity College, Dublin, Ireland) for her generous gift of S. aureus strain (8325-4), Dr. M. Merten (Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, Vandoeuvre-Les-Nancy, France) for the MM-39 cell line, C. Macet and Prof. P. N'Guyen (Laboratoire d'Hématologie, CHU Reims, France) for the T cells obtained by elutriation, and Drs. D. Marsh and A. Belaaouaj for critically reviewing the manuscript.


   Footnotes
 
This work was supported by Association Vaincre la Mucoviscdose (AVLM). E.S. and A.D. are supported by AVLM.

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0191OC on November 11, 2005

Received in original form May 18, 2005

Accepted in final form September 13, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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